The effect of excipients on pharmacokinetic parameters of parenteral drugs

Inauguraldissertation

zur

Erlangung der Würde eines Doktors der Philosophie vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel

von

Barbara Egger-Heigold

aus Grindelwald (BE), Littau (LU) und Plasselb (FR)

Basel, 2005

II

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Hans Leuenberger PD Dr. Georgios Imanidis Dr. Bruno Galli Basel, den 20. September 2005

Prof. Dr. Hans-Jakob Wirz Dekan

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Contents

Summary ................................................................................................................... V

Abbreviations.......................................................................................................... VII

1 Introduction ............................................................................................ 1

1.1 The physiology of blood ........................................................................... 1 1.1.1 The blood cells ............................................................................. 1 1.1.2 Plasma ......................................................................................... 1

1.2 In vitro methods to investigate blood binding parameters ........................ 2 1.2.1 Blood distribution method ............................................................. 3 1.2.2 Protein binding methods............................................................... 3

1.3 Characterization of drug candidates ........................................................ 4 1.3.1 Physicochemical properties.......................................................... 4 1.3.2 Pharmacokinetic parameters........................................................ 5 1.3.3 New trends in characterizing drug candidates.............................. 6

1.4 Strategies and administration of intravenous formulations....................... 7

1.5 Effect of excipients on pharmacokinetic parameters in blood .................. 9 1.5.1 Cremophor EL .............................................................................. 9 1.5.2 Cyclodextrins.............................................................................. 10 1.5.3 Tween 80.................................................................................... 10 1.5.4 Other excipients.......................................................................... 11 1.5.5 Nanoparticles.............................................................................. 11

1.6 Objectives and specific aims.................................................................. 12

2 Selection and experimental procedure .............................................. 13

2.1 Excipients and model compounds ......................................................... 13

2.2 Experimental setup ................................................................................ 15

3 Materials and methods ........................................................................ 17

3.1 Chemicals .............................................................................................. 17

3.2 Blood and plasma sources..................................................................... 17

3.3 In vitro studies........................................................................................ 17 3.3.1 Preparation of test solutions ....................................................... 17 3.3.2 Hemolytic activity........................................................................ 18 3.3.3 Blood distribution........................................................................ 18 3.3.4 Plasma protein binding ............................................................... 19 3.3.5 Determination of protein concentration....................................... 20

IV

3.4 In vivo studies ........................................................................................ 20 3.4.1 Experimental animals ................................................................. 20 3.4.2 Drug administration and sample collection ................................. 20 3.4.3 Bladder catheterization and urine collection ............................... 21 3.4.4 Ex vivo protein binding ............................................................... 21

3.5 Measurement of the radioactivity ........................................................... 21

3.6 Determination of parent drug ................................................................. 22

3.7 Data analysis ......................................................................................... 22

3.8 Pharmacokinetic analysis....................................................................... 23

4 Results and discussions..................................................................... 24

4.1 Hemolytic activity of excipients .............................................................. 24

4.2 Impact of the hematocrit on blood partition parameters ......................... 25

4.3 Major binding proteins of model compounds.......................................... 26

4.4 The impact of Vitamin E TPGS on COM1 in rat ..................................... 27

4.5 The impact of Vitamin E TPGS on COM2 in mouse .............................. 29

4.6 The impact of hydroxypropyl-β-cyclodextrin on COM3 in rat ................. 33

4.7 The impact of Cremophor EL on COM4 in rat........................................ 39

4.8 The impact of Solutol HS 15 on COM5 in rat ......................................... 42

5 General discussion and conclusions................................................. 47

6 Outlook ................................................................................................. 54

7 References............................................................................................ 56

8 Appendix............................................................................................... 64

9 Acknowledgments ............................................................................... 81

10 Curriculum Vitae .................................................................................. 82

V

Summary

In the pharmaceutical industry, the main goal of early phase in vivo studies is to assess pharmacokinetic properties of a compound in laboratory animals. These data provide a basis for selecting and optimizing drug candidates. However, formulation scientists face considerable challenges in finding intravenous preparations for first animal experiments. A common problem is the solubilization of lipophilic and sparingly water-soluble compounds. The search for suitable delivery vehicles often takes place under little compound availability, incomplete physicochemical property characterization, and time constraints. In addition, many experiments have recently generated distinct evidence about the impact of formulation vehicles on the drug pharmacokinetics by affecting transporters, metabolic enzymes, and distribution processes. Consequently, drug-excipient interactions are important to consider in the development of parenteral formulations intended for the proper evaluation of animal pharmacokinetics in vivo. Gaining a better understanding of potential interactions between drug and formulation in preclinical settings may play a crucial role in clinical and commercial phases of development as well.

So far, little is known about drug-excipient interactions occurring in blood, especially following iv administration of low dosed compounds ( 1h) in various species (mouse, rat, dog, and human), whereas TPGS at 0.1% showed no hemolysis under same conditions. Nevertheless, TPGS (0.5%) was used in the non-hemolytic time range for further investigations. The concentration of all excipients was set at 0.5% in test systems which is within the relevant range following intravenous dosing in animals.

VI

In vitro, CEL, HP-β-CyD, Solutol, and TPGS influenced clearly the plasma protein binding and the distribution between blood cells and plasma of model compounds in mice (COM2) or rats (COM1, COM3, COM4, COM5). The addition of TPGS to incubations increased the distributed fraction of COM1 and COM2 in plasma with a concomitant decrease of drug unbound in plasma. Formulating COM4 in CEL and COM5 in Solutol lowered the protein binding, and the higher drug fraction unbound in plasma was associated with enhanced partitioning into blood cells. The presence of HP-β-CyD reduced both the uptake of COM3 into blood cells and the binding to plasma proteins.

To assess the correlation between the in vitro findings and the in vivo situation, pharmacokinetics and tissue distribution were determined up to 1 h (within PET scan times) after an intravenous bolus injection of model compounds in formulations based on excipients or none (control) to animals, using in each case the excipient with the most pronounced interactions detected in vitro. Injection preparations contained the excipient to yield estimated blood concentrations of about 0.5%, similar to those used in the in vitro experiments. COM2 formulated in TPGS caused a higher accumulation of parent drug and metabolites in plasma without affecting tissue levels in mice. Administering COM3 in HP-β-CyD altered the disposition of COM3 characterized by a lower binding to plasma proteins, decreased drug levels in the systemic circulation and skin, and a higher amount of unchanged drug in the urine. COM4 formulated in CEL resulted in a higher drug fraction unbound in plasma which had no impact on the pharmacokinetics and tissue distribution. The use of Solutol for COM5 application in rats was associated with decreased protein binding, longer persistence in the circulation, and higher concentrations in muscle and skin. Although TPGS induced a slight shift in the pharmacokinetic parameters of COM1 in rats, the compound turned out to be an inappropriate model compound due to its very rapid metabolism and elimination under in vivo conditions.

These in vitro and in vivo findings demonstrated that commonly used excipients have a substantial potential for drug-excipient interactions in blood by altering protein binding and blood cell/plasma distribution which can influence the tissue distribution and elimination within the first hour after dosing. As a result, the formulation vehicle can be an important determinant for the disposition of low dosed compounds administered intravenously in animals. Moreover, results indicate a direct correlation of the excipient effect under in vitro and in vivo conditions. Therefore, blood distribution and plasma protein binding data generated in vitro seem to be appropriate to reveal potential drug-excipient interactions, thereby providing helpful information to improve the rational approach and strategy in the development of parenteral formulations at the preclinical stage. A better insight into the contribution of excipients to drug pharmacokinetics suggests also new possibilities of targeting different blood compartments and tissues by selecting the appropriate excipient. Such investigations should be considered to develop formulations suitable for intravenous administration of PET ligands where sub-therapeutic doses and short scanning times are used.

VII

Abbreviations

AGP α1-acid glycoprotein AUC Area under the drug concentration-time curve BCPR Ratio of concentration in blood cells to that in plasma, no units BPR Ratio of concentration in blood to that in plasma, no units C0 Initial plasma concentration at time zero CB Concentration of drug in blood CBC Concentration of drug in blood cells CEL Cremophor EL CP Concentration of drug in plasma EtOH Ethanol FP Drug fraction distributed in plasma, % fu Fraction of unbound to total drug concentrations in plasma, % funchanged AUC ratio of parent drug to that of total radioactivity, % Glu 5% aqueous solution of glucose H Hematocrit HDL High density lipoprotein HP-β-CyD Hydroxypropyl-β-cyclodextrin im Intramuscular iv Intravenous k Rate constant, h-1 KP Distribution ratio of drug between tissue and blood/plasma, no units LC-RID Liquid chromatography-reverse isotope dilution LDL Low density lipoprotein LOQ Level of quantification LSC Liquid scintillation counting nd Not determined PEG 200 Polyethylene glycol 200 PET Positron emission tomography SD Standard deviation Solutol Solutol HS 15 TPGS D-α-tocopheryl polyethylene glycol 1000 succinate t1/2 Half-life, h V0 Volume of distribution based on initial drug concentration in plasma, L VLDL Very low density lipoprotein ρ Ratio of concentration in blood cells to that unbound in plasma, no units

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1 Introduction

1.1 The physiology of blood

Blood is composed of cellular elements suspended in the plasma, an aqueous fluid in which solids are dissolved. Table 1-1 summarizes the main blood constitution of different laboratory animal species and humans. The normal range can vary, depending mainly on genetic and environmental factors and methods handling.

Table 1-1 Normative data for laboratory animals and humans

Mouse (1,2) Rat (1,3,4) Human (5) Sex Male Male Male Strain OF1 Wistar Body weight (kg) 0.030 0.250 70 Whole blood (ml/100 g) 7.2 (6.3-8.0) 7.2 ± 0.2 7.1 ± 0.6 Plasma (ml/100 g) 3.2 3.9 ± 0.1 4.4 ± 0.5

Total plasma proteins (g/100 mL) 5.4 ± 0.2 5.7 ± 0.5 7.5 ± 0.4

Albumin (% plasma proteins) 61 ± 1 48 ± 3 62 ± 3 α1 globulin (% plasma proteins) 17 ± 2 4 ± 1 α2 globulin (% plasma proteins)

12 ± 1 (α globulin) 10 ± 2 9 ± 1

β1 globulin (% plasma proteins) β2 globulin (% plasma proteins)

20 ± 1 (β globulin)

19 ± 1 (β globulin)

11 ± 2 (β globulin)

γ globulin (% plasma proteins) 7 ± 1 6 ± 1 15 ± 2 Blood cells Hematocrit (%) 43 ± 3 46 ± 2 44 ± 2 Red blood cells (x106 cells/µL) 9 ± 1 7 ± 1 5 ± 1 White cells (x103 cells/µL) 4 ± 2 6 ± 2 7 ± 1 Platelets (x106 cells/µL) 1.3 ± 0.4 1.2 ± 0.2 0.3 ± 0.1

1.1.1 The blood cells

The different specialized cells found in blood are white blood cells (leukocytes), red blood cells (erythrocytes) and platelets (thrombocytes). Of these, the erythrocytes are the most numerous and compose about one-half of the circulating blood volume. By carrying hemoglobin in the circulation, the red blood cells supply O2 to tissues and remove CO2. Leukocytes are classified as granulocytes (further classification in neutrophils, eosinophils, and basophils), lymphocytes, and monocytes. Acting together, these cells provide the body with a powerful defense against tumors, viral, bacterial, and parasitic infections. Compared to the other blood cells, the platelets are much smaller and aid in hemostasis by their primary function in blood clotting. Furthermore, blood cells can play a key role in binding and transporting of drugs in the circulation, thereby contributing to their pharmacokinetic and pharmacological characteristics (6,7).

1.1.2 Plasma

The plasma, the liquid portion of the blood, is a complex fluid composed of water (approximately 90%) and a large number of ions, inorganic molecules, and organic molecules in solution. These dissolved substances, primarily proteins, are in transit to

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various parts of the body or aid in the transport of other substances. The plasma proteins consist of albumin, globulin, and fibrinogen fractions, which can be separated by electrophoresis. Electrophoretic separation followed by immunoprecipitation (immunoelectrophoresis) results in a further division of the proteins. If whole blood is allowed to clot and the clot is removed, the remaining fluid is called serum and has essentially the same composition as plasma except for the removed fibrinogen and few clotting factors (II, V, and VIII). Table 1-2 lists the main protein fractions with their main characteristics. The table also indicates that a large number of drugs associate with proteins within the bloodstream. Albumin is the major drug-binding plasma protein (8) followed by alpha 1-acid glycoprotein as the next important one (9). In recent years, studies have shown, that lipoproteins are also substantially involved in the binding/transport of drugs in the blood compartment (10). So far, γ-globulins play only a marginal role in plasma binding of drugs.

Table 1-2 Proteins in human plasma

Physiological Function

Albumin PrealbuminAlbumin

α1 globulin α1-acid glycoprotein Uncertain (acute phase protein)

α1-lipoprotein ("high Transporter Lipidsdensity lipoproteins")

α2 globulin Ceruloplasmin Transporter Copperα2-Macroglobulin Enzyme inhibitor Serum endoproteasesα2-Haptoglobin Binding and carrier protein Cell-free hemoglobin

β globulin Transferrin Transporter Ironβ-lipoprotein ("low Transporterdensity lipoproteins")Fibrinogen Precursor to fibrin in hemostasis

γ globulin IgG, IgA, IgM, IgE Antigen Few basic compounds

Electro-phoresis

Immuno-electrophoresis

Protein fraction Binding characteristics

Drugs Endogenous entities

Lipoproteins: mainly lipophilic neutral and basic compounds

Humoral immunity (antibodies/immunoglobulins)

Lipids (mainly cholesterol)

Binding and carrier protein, osmotic regulator

Hormones, amino acids, steroids, vitamins, fatty acids

Mainly basic and neutral compounds

Mainly acidic, but also basic and neutral compounds

Lipoproteins: mainly lipophilic neutral and basic compounds

1.2 In vitro methods to investigate blood binding parameters

The investigation of the partitioning of a drug in the blood compartment is essential in predicting its pharmacokinetic/-dynamic profile. In general, the unbound concentration of a drug in blood reflects more accurately pharmacological effects of the drug than its total concentration in blood (bound + unbound), because only the drug unbound to blood components is able to diffuse through the membranes and then reach the target organ (11). Furthermore, the binding to plasma proteins also relates to the volume of distribution and the clearance of the drug. For instance, many experimental and clinical studies have generated substantial evidence summarized by Akhlaghi (12), that the unbound fraction of cyclosporin in plasma correlates more closely with pharmacodynamic and pharmacokinetic characteristics of cyclosporin than its total blood concentration. Therefore, determination of extent and rate of blood/plasma distribution and plasma protein binding of a drug is important in both the discovery and clinical phases of drug development.

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1.2.1 Blood distribution method

The rate and extent of blood/plasma distribution of drugs is determined in vitro in spiked whole blood. The experiments are performed under controlled physiological conditions (pH 7.4, 37°C, gently shaken) to reflect the in vivo situation over the entire clinically relevant concentration range of the drug. Time samples are taken and centrifuged. Subsequently, drug concentrations in blood and plasma are determined to calculate the time required to reach equilibrium. The extent of blood/plasma and blood cell/plasma distribution derives from measured concentrations in blood and plasma and can be expressed with distribution parameters like FP, BPR, and BCPR. BPR depends on the hematocrit of the whole blood used in the determination, whereas BCPR is independent of the hematocrit value.

1.2.2 Protein binding methods

Various methods are available for the determination of free drug concentration and protein-drug binding fraction in plasma (13,14,15), including conventional separation methods summarized in Table 1-3. However, the routinely used methods like ultrafiltration or equilibrium dialysis are limited in the case of lipophilic drugs due to their nonspecific adsorption to ultrafiltration device or to the dialysis membrane. Along with a trend to more lipophilic compounds observed in the pharmaceutical industry in recent years (16), these adsorption problems are expected to increase. As a result, ongoing method modifications and new methods are needed to overcome these difficulties. Overall, the selection of the method of binding assay depends upon the aim of the study and the physicochemical properties of the particular test compound including its formulation.

The ratio of bound and total drug concentrations in plasma expresses the degree of drug binding to plasma proteins and ranges between values of 0 and 1. Based on these values, drugs can be classified into very highly bound (>0.95), highly bound (>0.90), poorly bound (

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Table 1-3 Conventional methods for determination of plasma protein binding

Method Principle Advantages Disadvantages

Ultrafiltration

Ultracentrifugation

Gel filtration Adoptable for lipophilic drugs, automatable, binding differences detectable (e.g. affinity)

Complex handling, time consuming

Time consuming, expensive equipment, false estimation of free fraction by physical phenomena (e.g. sedimentation, back diffusion), protein contamination of free drug layer

Separation by size exclusion and affinity of column

Separation by centrifugation at high speed in absence of a membrane

No membrane effects, "natural environment", no dilution problems, adoptable for lipophilic and high MW drugs, evaluation of lipoprotein binding

Equilibrium dialysis (reference method)

Sample dilution, volume shifts, Donnan effects, nonspecific adsorption, sieve effect, time consuming, unsuitable for unstable drugs

Separation by filtration through a semipermeable membrane with defined molecular weight cutoffs accelerated by centrifugation or positive pressure (N2 gas, syringe)

Equilibrium establishment between two compartments separated by semipermeable membrane with defined molecular weight cutoffs

Physiological conditions, universal binding method

Simply applicable, short analysis time, simple commercially available kits, no volume shifts, no dilution effects

Donnan effects, nonspecific adsorption, binding equilibrium changes during separation process, small amount for analysis, sieve effect

1.3 Characterization of drug candidates

Successful candidates in drug development must have proper physicochemical properties in addition to acceptable pharmacokinetics, efficacy, and safety profiles. As a result, a clear understanding of compound characteristics and their correlations are helpful to rank and sort out unsuitable compounds in drug research (17,18).

1.3.1 Physicochemical properties

The chemical structure of a drug candidate is used in both predicting the pharmacology and selecting formulation strategies. Table 1-4 shows physico-chemical parameters, which are critical for in vivo drug action.

The molecular weight (MW) indicates roughly the size of a chemical entity and is connected to its membrane permeability, namely to the intestinal and brain penetration (16,19).

LogP, the octanol-water partition coefficient, has been widely accepted as a measure of molecular lipophilicity. Lipophilicity affects both the pharmacokinetic and pharmacodynamic behavior of drug molecules (20,21). LogP considers the molecule in its neutral state (neutral substance or ionizable substance in its neutral form), whereas logD reflects the pH-dependent distribution coefficient, consequently taking the ionization of molecules into account. If logP and pKa of a compound are known, logD can be calculated at any pH (21).

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The aqueous solubility (LogS) is closely related to drug stability, liberation, and absorption by passive diffusion, thereby playing a key role in its bioavailability (22). Causes for poor solubility are mainly excessive lipophilicity and crystal packing issue (23,24,25,26). The solubility of nonionic molecules is pH independent, while molecules with ionizable groups show pH dependent solubility. Acid drugs have higher solubility at pH higher than pKa and basic drugs at pH lower than pKa due to better solubility of ionic species as compared to the neutral species. The acid-basic character accounts also for crossing the blood-brain barrier (27).

The polar surface area (PSA) of a molecule is a useful parameter for predicting drug transport properties. PSA is the sum of the molecular surface (either van der Waals or solvent-accessible) that arises from polar atoms, usually N, O, N-H, and O-H atoms. Some scientists also include sulphur and phosphor and attached hydrogens as polar atoms. The PSA of a compound is also closely related to its hydrogen bond accepting and donating ability which can be responsible for interactions with active efflux pumps (28,29). PSA has been shown to correlate well with blood-brain distribution (27,30,31), intestinal absorption (32,33,34,35,36,37), and oral bioavailability (38) of compounds.

Table 1-4 Physicochemical parameters

Parameter Description Predictor Optimal value

MW Molecular weight Size, Permeability < 500< 450 (BBP)

LogP < 5

LogS Hydrophilicity > 20 µg/mL

pKa Acid-base character

PSA Polar surface area < 140 Å< 80 Å (BBP)

Negative logarithm of the acid-base dissociation constant

Acids >4 and bases

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AUC is the total area under the curve that describes the concentration of drug in blood or plasma as a function of time. AUC represents the drug exposure and is calculated by the trapezoidal rule.

The volume of distribution (V0) relates the amount of drug in the body to the concentration of drug in the blood or plasma, depending upon the fluid measured. This volume does not necessarily refer to an identifiable physiological volume, but merely to the fluid volume that would be required to contain all of the drug in the body at the same concentration as in the blood or plasma:

o

iv

C

DoseV =0

C0 represents the blood or plasma concentration at time zero and is determined by extrapolation to zero time of the linear plot of concentration vs. time in semilogarithmic scale.

The half-life (t1/2) is the time it takes for the blood or plasma concentration or the amount of drug in the body to be reduced by 50%:

kkt

693.02ln2/1 == ,

where k is the elimination rate constant, which can be calculated by the slope of the best-fit line to a semilogarithmic plot of the concentration over time. The relationship of t1/2 to both clearance and volume of distribution is given by:

V

CLk =

1.3.3 New trends in characterizing drug candidates

Before conducting clinical trials in humans, preclinical testing is carried out to discover the pharmacology, ADME (adsorption, distribution, metabolism, and excretion), and toxicology of a new drug candidate (39). Appropriate pharmacokinetics and a good balance between drug efficacy and safety contribute mainly to an efficient and effective drug development. However, these factors are the major hurdles in development which primarily cause increased costs and failure rate of candidates. Thus, pharmaceutical industry needs new concepts able to speed and improve activities and decision-making in drug development (40,41). In this context, microdosing, biomarkers, and PET ligands can help to prioritize resources and optimize drug selection in development. In many cases, these approaches deal with compound concentrations ranging from sub-therapeutic to low pharmacological levels, and thus information obtained from these techniques must reflect correctly the conditions at therapeutic doses, including interactions with macromolecules like enzymes, transporters, and proteins. In the end, a successful integration requires a profound understanding of strengths and limitations of these new concepts.

The administration of a low dosed (microdosed) drug candidate to humans was proposed to obtain human pharmacokinetic data before conducting Phase I trial (42). A microdose is one-hundredth of the proposed pharmacological dose determined from animal and/or in vitro models, or a dose up to 100 µg, whichever is the smaller (43). Human microdosing uses labeled agents administered mostly intravenously, and their fate in vivo is recorded by positron emission tomography combined with accelerator mass spectrometry or nuclear magnetic resonance (43,44). With this new

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strategy of microdosing, drug information regarding human kinetics will be available along with preclinical data and can be useful for the acceptance or rejection of a candidate at an early stage.

A biomarker is an indicator of a normal biological or pathophysiological process or a therapeutic response (45). Biomarkers help to select the most sensitive drugs in all phases of drug development (46) by providing data of pharmacological response, dosing regimen, and risk-benefit assessment. Therefore, efforts are moving rapidly forward to achieve strong predictive biomarkers which could be used for diagnostic and therapeutical purposes (47).

PET tracers labeled with short-lived radionuclides (e.g. 11C, 18F, 124I) are used as molecular probes of physiology and pathophysiology in animals and humans. These labeled compounds are administered mostly intravenously at 600 MBq to humans which corresponds to 6-20 nmol (3-10 µg assuming a MW of 500) (48). To achieve the same imaging quality in animals, roughly the same total amount of radiopharmaceutical must be given to animals as to a human subject (49).

1.4 Strategies and administration of intravenous formulations

In the pharmaceutical industry, formulation scientists have faced growing challenges in recent years as a result of new drug candidates characterized as being more lipophilic, hydrophobic, and water-insoluble, particularly candidates originated from leads associated with combinatorial chemistry and high-throughput screening (16,24). In addition, timelines and resources are very limited to develop an optimized formulation and thus the search for a suitable dosing vehicle intended for activities in preclinical research represents a challenging task for the formulators (50). Ideally, it is best to select and use solubilizers that would maximize the solubility of the compound and could be applied for all preclinical settings. Moreover, the solubilizing agents should not influence the intrinsic pharmacokinetic characteristics of the compound being evaluated (except the interaction is well understood), which would lead to misinterpretation of the pharmacological response (51). Strategies for solubilization of intravenous drugs are summarized in Table 1-5 and well exemplified by the formulation approaches for the anticancer agent Paclitaxel (52).

Usually, the first step is to check the solubility of the compound in an aqueous dosing vehicle at physiological pH and osmolarity. If the target concentration cannot be achieved with this approach and the drug molecule is ionizable, adjustment of the pH to non-physiological values can be suitable to increase water solubility (pKa must be sufficiently away from the formulation pH). Non-electrolytes are insensitive to pH modification. The next approach most frequently tried is the addition of water-miscible organic solvents (cosolvents) and the use of surfactants or complexing agents. To reach the required dose, combination of these methods is often used. Dispersal systems are other techniques, but they may be difficult, costly, and time-consuming due to biological and technical complexity, e.g. liposomes (53).

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Table 1-5 Intravenous formulation approaches

Approach Examples Administered in commercial productsa Potential drawbacks

NaCl 0.9% (w/v), pH 2-12 (bolus), pH 2-10 (infusion) PrecipitationGlucose 5% (w/v) → preferred range pH 4-9 PainStrong acids/bases (HCl, NaOH),Buffers (tartarte, phosphate)

Cosolvents Propylene glycol ≤ 68% (bolus), ≤ 6% (infusion) Precipitation Ethanol ≤ 20% (bolus), ≤ 10% (infusion) Irritation/PainPolyethylene glycol 300 ≤ 50% (bolus) HemolysisPolyethylene glycol 400 ≤ 9% (bolus) Impact on PK profile

Surfactants Cremophor EL ≤ 10% (infusion) ditoTween 80 ≤ 0.4% (bolus), ≤ 2% (infusion)Solutol HS 15 50%

Complexing agents Hydroxypropyl-β-cyclodextrin 20% (infusion) ditoDispersal systems: Impact on PK profile

Emulsionb/Microemulsionc Water with 10-20% oil (fatty acids + lecithin + glycerol) Sustained releaseLiposomes Water with phospholipids (5-20 mg/mL) + isotonicifier + buffer ± cholesterol Instability

Nanosuspensiond Water with stabilizer not yet marketede Slow dissolution

Aqueous solution at physiological osmolarity and pH / or with pH adjustment

a(54), b(55), c(56), d(57), e(58)

For compounds administered intravenously to animals, the dose volume, viscosity

of injection material, speed of injection, and species are important factors to consider in addition to formulation properties including additives, solubility, and stability (Table 1-6) (59). A compound can be given over a short period of ≤1 min (bolus injection), 5-10 min (slow injection), and longer time period (intravenous infusion). Rapid injections require the dose to be compatible with blood and not too viscous, and the rate of injection is suggested not to exceed 3 mL/min for rodents. Depending on study objectives and compound solubility in an acceptable formulation, a larger volume may be needed to be given to animals to accomplish requirements. Regarding the formulation, aqueous solutions or simple systems containing cosolvent, surfactant, or complexing agent are recommended for animal investigations at early stage in development due to easy handling and characterization. For excipient selection, consideration should be given for toxic and biological effects, interferences with the drug compound, and suitability for clinical use (Table 1-5). Injectable excipients preferred for dosing in animals are: ethanol, propylene glycol, low molecular weight polyethylene glycols, Cremophor EL, Tween 80, and cyclodextrins.

Table 1-6 Dose volumes and rates for intravenous administration(59)

Species Bolus injection Slow injection Infusion

Volume Rate Volume Rate Time Volume Rat

(mL/kg) (mL/min) (mL/kg) (mL/min) (h) (mL/kg/d) (mL/kg/h) Mouse 5 3 max. 25 3 4 24 96 4 Rat 5 3 max. 20 3 4 20 5 24 60 2.5

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1.5 Effect of excipients on pharmacokinetic parameters in blood

Over the last years, more attention has been paid to the extensive investigation of formulation vehicles as biologically and pharmacologically active compounds. The main stages in which pharmaceutical excipients can interact and hence may modulate the properties of an administered drug-agent are transporter, enzyme, and distribution process in the systemic circulation (e.g. plasma protein binding). The effect of excipients on transporter activity has been studied intensively, namely for P-glycoprotein (60,61,62,63,64,65,66,67,68), multidrug resistance-associated protein (69,70) and peptide transporter (71). It is interesting to say that particularly nonionic surfactants effectively inhibit transporters. In contrast, up to this day little is known about drug-excipient interactions at the level of cytochrome-mediated metabolism (63,72,73,74) and blood distribution (see below). The biological and pharmacological properties of excipients with a focus on the central blood compartment will be reviewed in the following paragraphs.

1.5.1 Cremophor EL

The amphiphilic polyethoxylated castor oil derivative Cremophor EL (CEL) is one of the most frequently used surface-active formulation ingredients in parenteral dosage forms. As early as 1977 lipoprotein alterations were observed in patients receiving miconazole therapy (75) which was caused only by CEL, both in vitro and in vivo (76,77,78). Extended studies revealed later on that CEL has a destructive effect on HDL resulting in a shift of the electrophoretic and density gradient HDL to LDL (79,80,81,82). Furthermore, several hydrophobic anti-tumor agents, tin etiopurpurin (83,84), C8KC (85) and Taxol (81,82), showed strong affinity for these lipoprotein dissociation products inducing changes in plasma protein binding, potentially affecting pharmacokinetics.

Various animal studies demonstrated (85,86,87,88,89,90,91,110) that CEL modifies the pharmacokinetic behavior of drugs after intravenous administration, like paclitaxel (Taxol), C8KC, and cyclosporin. The most common observation was a substantial increase in the area under the plasma concentration-time curve and in peak plasma concentration of studied agent with a reduction in the clearance, as was first described for paclitaxel in a mouse model (91). The drug-CEL interactions were supposed to be caused not only by altered protein binding characteristics (82), but also by altered hepatobiliary secretion (92) and endogenous P-glycoprotein-mediated biliary excretion (93). However, the very small volume of distribution of CEL, approximately equal to the volume of the central blood compartment, suggests that the observed interference occurs in the central blood compartment. This hypothesis was confirmed by studies recently published (94,95). The main finding was a profound alteration of cellular partitioning and blood/plasma concentration ratio of paclitaxel in a CEL concentration-dependent manner as a result of an entrapment of the compound into micelles formed by CEL (96). Consequently, the free drug fraction available for distribution was reduced. This effect was also observed in the absence of plasma proteins, pointing at contributing factors other than altered protein binding and increased affinity of paclitaxel for CEL-induced lipoprotein degradation products (81,82).

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For the purpose of finding out a potential paclitaxel delivery vehicle with an ideal profile, the investigation of several delivery vehicles based on the chemical structures of CEL and Tween 80 led to alteration of blood distribution of paclitaxel in presence of all tested vehicles (97). Different formulation approaches such as liposomes and poloxamer-micelles affected the pharmacokinetics of paclitaxel in mice as compared to the CEL-containing formulation (98). In contrast, paclitaxel administered in a solvent-free formulation in a clinical study showed a higher efficacy combined with reduced adverse effects compared to the drug delivered in a solution of CEL (99).

Overall, numerous investigations have shown that CEL can play a pivotal role in the pharmacological behavior of the formulated drugs. In addition, several drug-drug interactions are reported on agents administered intravenously in conjunction with CEL-containing formulation of other compounds, namely paclitaxel (100,101,102,103), cyclosporin (104,105,106,107), and valspodar (108,109). Most likely, the presence of CEL in drug formulations contributes to the observed pharmacokinetic interactions. Indeed, recent experiments revealed a substantial increase of plasma concentrations of cyclosporin after an additional injection of another drug preparation containing CEL (110).

1.5.2 Cyclodextrins

Cyclodextrins are ring-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic interior (111). The interior cavity is capable of forming water-soluble complexes with many drugs. Due to the rapid release of a drug from the complex after administration in vivo, it is assumed that drug-cyclodextrin complexes do not affect the drug pharmacokinetics (112). However, if the drug is slowly or incompletely released from the complex, drug dosing as complexes in cyclodextrin can be critical.

The binding of drugs to plasma proteins was influenced in vitro in the presence of β-cyclodextrin (113) and hydroxypropyl-β-cyclodextrin (HP-β-CyD) (114,115). The intravenous administration of flurbiprofen in HP-β-CyD led to transient higher tissue concentrations in rats (114). Alterations in tissue distribution were also found for other drugs injected as cyclodextrin complexes either free in solution (116,117) or included into liposomes (118). Following iv dosing in HP-β-CyD, a higher amount of carbamazepine appeared in the urine compared to oral preparations (122). A similar trend was observed in dogs treated iv with either dexamethasone formulated in HP-β-CyD or as its phosphate prodrug (123). In addition, cyclodextrins interacts with endogenous lipids such as lipoproteins (119,120) and cholesterol (113,121).

1.5.3 Tween 80

As mentioned above for CEL, lipoprotein alteration induced by Tween 80 was observed (80). However, this effect was not confirmed in a further study (85). In patients receiving Tween 80 co-administered with etoposide, an increase of the volume of distribution and the clearance of doxorubicin was detected due to reduced plasma concentrations of doxorubicin during the early phase of the concentration-time profile (124). Lately, changes in the blood/plasma ratio of paclitaxel were described in the presence of Tween 80 and other solubilizers structurally related to Tween 80 (97). More recently, it was shown that Tween 80 has a concentration-dependent influence on the normal binding of docetaxel to serum proteins leading to changes in pharmacokinetics of docetaxel in vivo (125) although Tween 80 is

11

degradated rapidly by esterases in plasma (126). The mechanistic basis for altered plasma binding of docetaxel in the presence of Tween 80 still needs to be clarified.

1.5.4 Other excipients

To date, little is reported in the literature about the impact of Solutol HS 15 and Poloxamer 188 on blood distribution of drugs. An interference between Solutol HS 15 and the co-administered ketochlorin photosensitizer C8KC was suggested by Woodburn (127). The similar half-lives of Solutol HS 15 and the sensitizer found in mice indicate the correlation of the persistence of C8KC in plasma with that of the vehicle. Further, recent plasma protein binding interaction studies demonstrated an apparent increase in the unbound fraction of propranolol in combination with Poloxamer 188 (128). Also the administration of compounds formulated in mixed micelles can alter the protein binding (129). Most notably compounds binding with high affinity but low capacity to α1-acid glycoprotein showed free fractions increased by 50 to 85%. Moreover, blood protein interactions can occur with dosing vehicles like liposomes (130), thereby affecting maybe the fate of co-administered drugs in blood and body (131).

1.5.5 Nanoparticles

Methyl methacrylate nanoparticles of 130 nm in size suspended in different concentrations (0.1-5%) of Tween 80 or poloxamine 908 exhibited prolonged circulation time with altered tissue concentrations as compared to uncoated nanoparticles (132). Extended blood circulation time was also found for polystyrene nanoparticels (40-137 nm) coated with poloxamer 407 (133).

12

1.6 Objectives and specific aims

The main purpose of this thesis was to investigate in vitro drug-excipient interactions in blood and to assess the implications of the in vitro findings both for the in vivo situation and the formulation strategy. Compounds in drug development at Novartis were chosen as model substances and dosed at concentrations ranging from sub-therapeutic to low pharmacological levels. Excipients commonly used in formulations were selected, including CEL, HP-β-CyD, Solutol, PEG 200, and TPGS. The following specific aims of the thesis were: 1. To collect and use available compound information, including physicochemical

properties and pharmacokinetics, to select appropriate model substances with as many different properties as possible

2. To determine the hemolytic activity of selected excipients to rule out any changes of blood distribution caused by hemolysis

3. To explore in vitro possible effects of selected excipients in the blood, with special emphasis on the blood distribution and plasma protein binding of model compounds

4. To identify the pharmacokinetic profile and tissue distribution of model compounds following single intravenous dosing in the presence and absence of selected excipients

5. To compare and relate pharmacokinetic outcomes to the in vitro findings, thereby assessing the impact of in vitro data for the in vivo situation and evaluating the in vitro-in vivo correlation

6. To generate criteria for optimizing delivery vehicle selection in drug research that allow reducing drug-excipient interactions and leading to more rational and selective drug formulations

7. To propose an intravenous formulation strategy based on the data generated by this research project to provide better candidate-tailored formulations in drug development

13

2 Selection and experimental procedure

2.1 Excipients and model compounds

Investigations involved five excipients along with five pharmacologically active compounds exhibiting different properties.

The excipients CEL, HP-β-CyD, Solutol, and PEG 200 were selected based upon their common use in intravenous formulations and their diversity of molecular structure and solubilization technique (Figure 2-1 and Table 2-1). CEL and Solutol are surface-active agents which increase the drug solubility by incorporation of the drug into a micellar structure. Whereas CEL exhibit a highly variable composition with the major hydrophobic component (~87%) identified as oxyethylated triglycerides of ricinoleic acid (Figure 2-1), Solutol consists of ~70% lipophilic polyglycol mono- and di-esters of 12-hydroxystearic acid and ~30% hydrophilic polyethylene glycol. HP-β-CyD is a cyclic (α-1,4)-linked oligosaccharide containing seven α-D-gluco-pyranose units (Figure 2-1) which form a relatively hydrophobic central cavity and a hydrophilic outer surface. The inclusion of a drug within the inner core of the complexing agent and the interaction of the outer core with water render the complex soluble. PEG 200 is often used as a cosolvent for improving solubility of preclinical compounds by interrupting the hydrogen structure of water (e.g. water exclusion) and lowering the dielectric constant of the solution. Although TPGS is exclusively known in oral formulations, it was chosen due to its chemical structure (benzyl ring) and drug interaction potential at the level of active transporters and metabolizing enzyme systems.

Cremophor EL

H2C(CH

2CH

2O)xOCO(CH

2)7CH CHCH

2CHOH(CH

2)5CH

3

HC(CH2CH

2O)yOCO(CH

2)7CH CHCHCH

2CHOH(CH

2)5CH

3

H2C(CH

2CH

2O)zOCO(CH

2)7CH CHCH

2CHOH(CH

2)5CH

3

primary constituent with x + y + z ~35

Solutol HS 15

CH3

(CH2)5

CH (CH2)10

C O CH2

CH2

OHn

OH O

Polyethylene glycol 200

O

HOH

n

Hydroxypropyl-β-cyclodextrin

O

ROCH2

O

ROOH

n

glucopyranose with R=CH2CH2OH or H and n=7

Vitamin E TPGS

O

O C CH2 CH2 C O

O O

CH3

CH3

OH n

CH3

CH3

CH (CH2)3

CH3

CH (CH2)3

CH(CH2)3

n=20-22

Figure 2-1 Chemical structures of selected excipients

14

Table 2-1 Properties of selected excipients

Excipient name Type Solubilization Use in iv Biological activity

approach formulation Cremophor EL Non-ionic Micelles Yes • Dyslipidaemia (polyoxyethylene surfactant (developmental • Inhibition of active castor oil derivatives) & commercial) transporters MW ~3000 CMC ≥0.09 mg/mL Hydroxypropyl-β- Oligomeric Complexation Yes • Lipid interactions cyclodextrin substance (developmental MW ~1600 & commercial) Solutol HS 15 Non-ionic Micelles Yes • Dyslipidaemia (polyethyleneglycol 660 surfactant (developmental • Inhibition of active 12-hydroxystearate) & commercial) transporters MW 960 • Inhibition of cyto- CMC ≥0.2 mg/mL chrome enzymes Polyethylene glycol 200 Oligomeric Cosolvent Yes MW ~200 substance (change of (developmental solution & commercial*) polarity) Vitamin E TPGS Non-ionic Micelles No • Inhibition of active (D-α-tocopheryl surfactant (oral use: transporters polyethylene glycol 1000 developmental • Inhibition of cyto- succinate) & commercial) chrome enzymes MW ~1513 CMC ≥0.2 mg/mL CMC: Critical micelle concentration, MW: Molecular weight, *: Higher molecular weight PEGs such as PEG 300 and 400

Drug candidates in development at Novartis were chosen as model compounds

regarding aqueous solubility, lipophilicity, membrane permeability, and blood cell/plasma distribution (Figure 2-2 and Table 2-2). COM2 and COM1 (base) are lipophilic and poorly water-soluble PET ligands which are used in sub-therapeutic doses, and COM2 distributes equally between plasma and whole blood. COM3 is much better water-soluble and is mainly located in the cellular fraction in blood. In contrast, COM4 with a low lipophilicity penetrates hardly into blood cells and distributes poorly into tissues. COM5 is a bigger molecule characterized by a high polar surface area, many H-bond acceptors, and a very low volume of distribution similar to that obtained for COM4.

COM1 COM2 COM3 COM4 COM5

N

N

O

R1

R2

O

N

O

N

N

S

R1

R2

N

O

N

R

NN

R2

R1

N

NO

S O

NH

O

O

NH

OR1

R2

R3

Figure 2-2 Chemical structures of model compounds

15

Table 2-2 Properties of model compounds

Physicochemical (PC) and pharmacokinetic (PK) data available at the time of selecting model compounds for investigating drug-excipient interactions in blood.

COM1 COM2 COM3 COM4 COM5

PET ligand PET ligand NCE NCE NCE

PC properties MW (g/mol) 240 410 295

16

study where animals received intravenously a single dose as a control formulation or solution containing the selected excipient. Control formulation were based on glucose 5% (COM1, COM3), saline (COM4, COM5), or blank plasma (COM2). To assure a fast and complete solubility of COM3 and COM5 in the control formulation, convenient excipients were added with in vitro binding parameters similar to those obtained for the in vitro reference. The concentration of model compounds in blood, plasma, and tissue were measured until 1 h after iv administration, thereby including the scanning time of PET ligands. Moreover, it is assumed if excipient-induced changes occur they should be detectable in this time period.

17

3 Materials and methods

3.1 Chemicals

COM1 and COM2 were supplied by the Neuroscience Research Department of Novartis (Basel, Switzerland). COM3 was obtained from the Novartis Institutes for BioMedical Research (Basel, Switzerland). 3H-radiolabeled COM1 (specific activity 11780 MBq/mg, >99%), COM2 (specific activity 2320 MBq/mg, >98%), and COM3 (base, specific activity 31.1 MBq/mg, >98%) were provided by the Isotope Laboratories of Novartis (Basel, Switzerland). 14C-radiolabeled COM3 used for investigation of renal excretion (2·HCl salt, specific activity 5.87 MBq/mg, >98%), COM4 (specific activity 5.85 MBq/mg, >98%), and COM5 (specific activity 3.3 MBq/mg, >98%) were provided by the Isotope Laboratories of Novartis (Basel, Switzerland).

The excipients, purchased by the Pharmaceutical and Analytical Development Department of Novartis (Basel, Switzerland), were: Cremophor EL (CEL; BASF), hydroxypropyl-β-cyclodextrin (HP-β-CyD; CERESTAR USA Inc.), polyethylene glycol 200 (PEG 200; Fluka), Solutol HS 15 (Solutol; BASF), and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS; Eastman). All other chemicals and reagents were of analytical grade or will be described separately in the methods section.

3.2 Blood and plasma sources

Fresh blood was obtained from healthy male species (n≥3) as follows: mice (albino OF1, Charles River Laboratories, L’Arbresle, France), rats (Wistar HAN IGS, Charles River Laboratories, Sulzfeld, Germany), dogs (Marshall beagles, Marshall Farm, NY, USA and Harlan France SARL, Gannat, France), and humans (drug-free blood donors, Blutspendezentrum SRK Basel, Switzerland). Pooled plasma (n≥3) was defrosted from storage at -20°C. Lithium heparin was used as an anticoagulant for all species.

3.3 In vitro studies

Test compounds in the in vitro samples excluding protein binding samples of COM2 were quantified by LSC due to no major degradation (>95%) under investigated conditions (146,147,148,149,150). Protein binding samples of COM2 were quantified by LC-RID due to instability after longer incubation (>2 h) and very low fraction unbound in plasma (

18

10-300 ng/mL (COM5). Excipient concentrations were kept constant at 0.5% in the test system except for COM1 (0.5-5% due to no alterations at 0.5%) and COM4 (0.01-1% due to its plasma fraction, see 2.2).

3.3.2 Hemolytic activity

The hemolytic activity of the excipients was assayed using a spectrophotometric method. CEL/EtOH 65:35 (v/v), EtOH, HP-β-CyD, PEG 200, Solutol, and TPGS were added to the appropriate volume of freshly prepared heparinised whole blood to obtain a final excipient concentration of 0.5%. To avoid hemolysis due to higher concentrations during the adding, the blood was partially centrifuged and the excipient was pipetted in the cell-free layer. By tapping the test tubes, the samples were immediately mixed. Two control tubes were prepared, one for spontaneous hemolysis (pure blood used as the reaction blank) and another for 100% hemolysis (total cell lysis induced by sodium dodecyl sulfate at a final blood concentration of 1%). Samples were incubated at 37°C. At certain points of time, aliquots were removed and centrifuged for 10 min at 3100 x g (37°C) to obtain plasma. The absorbance of hemoglobin in the supernatant (dilution with water 1:200) was measured at 405 nm (Emax precision microplate reader, Bucher Biotech, Basel, Switzerland). The degree of hemolysis due to the excipient activity was calculated according to

100(%)0100

0×

−

−

=

AA

AAHemoylsis e

where Ae is the absorbance of hemoglobin in the supernatant after incubation with excipient, A0 is the absorbance of hemoglobin in the supernatant of the reaction blank, and A100 is the absorbance of hemoglobin in the supernatant after total cell lysis with sodium dodecyl sulfate. Hemolytic activity was considered to have started when mean values were greater than 2% of hemolysis.

3.3.3 Blood distribution

Freshly prepared heparinised blood was used, and experiments were performed in triplicate both in the presence and absence of excipients. The hematocrit was determined using a hematocrit centrifuge and a hematocrit reader (Haemofuge Heraeus Sepatech, Germany). In order to reduce hemolysis, blood aliquots (1 mL) were partially centrifuged (500 x g for 2 min) before adding the test solution in the cell-free layer, followed by mixing immediately. Samples were incubated at 37°C. Time aliquots (1 mL) were removed and prepared for measuring radioactivity of the test compound in whole blood before centrifugation and in plasma after centrifugation for 10 min at 3100 x g (37°C) by LSC.

The fraction of the test compound in plasma (FP) was calculated according to

100)1(

(%) ×−×

=

B

P

PC

HCF

where CB and CP are the drug concentration in blood and plasma respectively, and H is the hematocrit value. The concentration in blood cells (CBC) was calculated as follows:

19

H

HCCC PBBC

)1( −×−=

and used for calculations of blood cell to plasma concentration ratio (BCPR: CBC/CP) and blood cell to unbound in plasma concentration ratio (ρ: CBC/(CP*fu)). fu is the drug fraction unbound in plasma determined by the appropriate protein binding method for each compound.

3.3.4 Plasma protein binding

Control experiments in phosphate buffered saline (PBS, Gibco, Paisley, Scotland) were carried out to assess the suitability of the methods described below for each test compound in the following order: ultrafiltration > dialysis > ultracentrifugation, with ultrafiltration being the first procedure. Control experiments indicated that ultrafiltration is a suitable method for COM1, COM3, COM4, and COM5 (free-permeation >0.75, recovery >85%) and ultracentrifugation for COM2 (no sedimentation after 6-h centrifugation, recovery >85%; ultrafiltration and dialysis showed insufficient recovery and free-permeation). Therefore, protein binding was determined by the ultrafiltration technique (COM1, COM3, COM4, and COM5) or the ultracentrifugation technique (COM2).

Ultrafiltration

Samples of spiked plasma were incubated at 37°C until binding equilibrium. Aliquots of 1 mL were introduced in prewarmed (37°C) Centrifree micropartition tubes (Amicon Inc., Beverly, MA, USA) and centrifuged for 10 min at 2000 x g (37°C). For the determination of the unbound drug fraction in plasma, concentrations of the test compound in ultrafiltrate and plasma were measured. The unbound fraction in plasma (fu) was calculated as follows: fu(%)=(CUF/CP)x100, where CUF and CP are the drug concentration in ultrafiltrate and in plasma, respectively.

Equilibrium dialysis

Test solution was added to plasma followed by mixing. Dialysis was carried out with 150 µL of this sample against an equal volume of phosphate-buffered saline (pH 7.2) in a 96-well micro-equilibrium dialysis block (HTDialysis LLC, Gales Ferry, CT, USA). Dialysis membranes with a 12000-14000 molecular weight cut-off were soaked in phosphate buffered saline (pH 7.2) before use. After establishment of the equilibrium, buffer solution aliquots, containing only unbound drug, and plasma aliquots, containing both bound and unbound drug, were analyzed for the test compound. The ratio of drug concentrations measured in the buffer and plasma after dialysis was taken as an estimate of unbound drug fraction in plasma.

Ultracentrifugation

Samples of spiked plasma were incubated at 37°C until binding equilibrium. Aliquots of 1 mL were transferred to polycarbonate centrifuge tubes (Beckman) and either centrifuged in a TLA 100.2 rotor in Beckman TL 100 centrifuge (200000 x g, 6 h, 37°C) or incubated for 6 h (37°C). After centrifugation, samples were separated into three layers according to density. A 80-µL aliquot of the middle layer (protein-free part/plasma water) was taken and analyzed for the test compound, representing the unbound concentration in plasma (CU). Total plasma concentration (CP) was

20

determined in incubated samples. The unbound drug fraction in plasma was calculated using CU/CP.

Determination of major binding protein

The affinity of test compounds to different plasma proteins was determined using the appropriate method for each compound. Purified human plasma proteins were dissolved in phosphate buffered saline (PBS, Gibco, Paisley, Scotland) at physiological concentrations as follows: albumin 40 g/L (≥96%, Sigma), α-acid glycoprotein 1 g/L (from Cohn Fraction VI, 99%, Sigma), γ-globulins 12 g/L (from Cohn Fraction II and III, Sigma), high density lipoprotein 3.9 g/L (>95%, Calbiochem), low density lipoprotein 3.6 g/L (>95%, Calbiochem), and very low density lipoprotein 1.3 g/L (>95%, Calbiochem). Test solution was added to protein solutions to obtain a compound concentration of 10 ng/mL (COM1, COM2) or 1000 ng/mL (COM3, COM4, COM5). After incubation at 37°C, separation of bound and unbound compound was achieved according methods. Ultrafiltration was performed by centrifugation for 10 min for samples containing albumin and γ-globulins and for 2 min for all other samples.

3.3.5 Determination of protein concentration

Protein concentration was measured by the method of Bradford (Coomassie blue protein assay) at 595 nm by using a Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany). The protein concentration was determined by using a calibration curve that was established with known concentrations of human serum albumin (≥96%, Sigma) ranging from 0 to 0.5 mg/mL. 10-µL aliquots of plasma (1:200 dilution) and plasma water were pipetted into microtiter plate wells. 200 µL dye reagent were added, and samples were mixed. After 1-h incubation at room temperature, absorbance was measured.

3.4 In vivo studies

Samples collected after intravenous administration of COM1, COM2, and COM3 were assayed for radioactivity by LSC and parent drug by LC-RID. COM4 and COM5 were quantified in all in vivo samples only by radioactivity measurements (LSC) since the radioactivity of both radiolabeled compounds reflects well the parent drug due to no major degradation at 1 h after intravenous administration in rats (151,152).

3.4.1 Experimental animals

Male Wistar rats (~250 g) and male OF1 mice (~30 g) were obtained from Charles River (Sulzfeld, Germany). All animals were housed in standard cages in a controlled environment maintained on an automatic 12-h lighting cycle at a temperature of 22°C according to institutional guidelines. The animals were given a standard chow and water ad libitum. The animals were used after having been starved overnight.

3.4.2 Drug administration and sample collection

All dosing solutions were prepared within 1 h prior to injection and stored at room temperature until use. Administration was performed by a single bolus injection into the femoral vein after animals had been lightly anesthetized by isoflurane (Forene®). Rats received [3H]COM1 at 4 µg/kg as solution (1 mL/kg) in glucose 5% containing

21

ethanol 1% (v/v) or TPGS 20% (w/v). Mice were injected a dose of 400 ng/kg of [3H]COM2 formulated as solution (5 mL/kg) in blank plasma (obtained by centrifugation of freshly drawn mouse blood) or in glucose 5% containing TPGS 10% (w/v). An iv dose of 1 µmol/kg radiolabeled COM3 (3H: 300 µg/kg, 14C: 370 µg/kg) in EtOH/PEG200/Glu 5:5:90 (v/v/v) or 40% HP-β-CyD (w/v) was injected to rats (1 mL/kg). [14C]COM4 was administered at 400 µg/kg in saline or 17% CEL (v/v) to rats (2 mL/kg). [14C]COM5 at 1 mg/kg in saline containing either ethanol 10% (v/v) or 17% Solutol (w/v) were injected to rats (2 mL/kg).

Using these injection preparations, excipient concentrations in blood may be estimated as about 0.3% (COM1), 0.5% (COM3, COM4, COM5), and 0.7% (COM2) in animals (~70 mL blood/kg). These concentrations were similar to those used in the in vitro experiments.

Samples were collected after drug administration at 0.08, 0.25, and 0.5 h for COM1 and at 0.08, 0.25, 0.5, and 1 h for COM2, COM3, COM4, and COM5. Animals (n=3 per time point) were sacrificed by isoflurane inhalation for sample collection. Blood samples were collected from the vena cava and transferred into tubes containing heparin (heparin-Na, B.Braun) as anticoagulant. Plasma samples were obtained by immediate centrifugation of blood samples at 3000 x g for 10 min. Tissues were excised, blotted dry, and weighed. Collected tissue comprised lung, heart, liver, kidney, fat, muscle, skin, and brain for COM1, COM2, COM3 and lung, muscle, and skin for COM4 and brain, muscle, and skin for COM5. All samples were immediately frozen and stored at -20°C until analysis. Tissue samples were homogenized before quantification.

3.4.3 Bladder catheterization and urine collection

The experiment was performed in situ under anesthetized rats. Animals (n=3/formulation) received im injections of ketamine hydrochloride at a dose of 50 mg/kg (100 mg/mL, 0.5 mL/kg) and are positioned on an isothermal heating pad prewarmed at 38°C. The abdomen was opened through a mid-line incision. A polyethylene tubing (Clay-Adams PE-50) was inserted into the dome of the bladder and held in place with a purse string suture. The formulation was injected into the surgically exposed femoral vein, and urine was collected at 0.5, 1, 1.5, and 2 h after dosing. All samples were frozen and stored at -20°C until analysis.

3.4.4 Ex vivo protein binding

Ex vivo protein binding was determined for COM1, COM3, COM4, and COM5 according to the in vitro procedure. Briefly, remaining plasma samples of each time point were pooled, and the unbound drug concentration in plasma was quantified using the ultrafiltration technique (see 3.3.4). After centrifugation, plasma and ultrafiltrate samples were assayed for radioactivity by LSC and parent drug by LC-RID.

3.5 Measurement of the radioactivity

Aliquots of blood, plasma, urine (25-50 µL) and homogenates (250 µL) were introduced into counting vials and solubilized in Biolute-S (Zinsser Analytic). Samples obtained from in vivo studies containing tritium-labeled drug were dried, and the residue was reconstituted in water before solubilization. To the blood samples,

22

hydrogen peroxide 30% was additionally added, and vials were gently swirled for several seconds and let stand for 30 min. After adjusting pH >7 by addition of hydrochloric acid 2 N, the vials were filled with scintillation cocktail (Irgasafe Plus, Zinsser Analytic), kept in the dark for 16 h, and measured in a Tri-Carb liquid scintillation spectrometer Model A2200 (Packard).

3.6 Determination of parent drug 3H-radiolabeled COM1, COM2, and COM3 were determined by a liquid

chromatography-reverse isotope dilution method (LC-RID). A sample aliquot (100-500 µL) and 200 µL water containing 5 µg (COM1, COM3) or 2 µg (COM2) non-radiolabeled test compound as internal standard was added to a glass tube. After further addition of 1 mL water, 100 µL Titrisol buffer (pH 4: COM1, COM2; pH 7: COM3), and 4 mL diethyl ether (COM1, COM2) or tert-butylmethylether (COM3), tubes were shaken for 30 min and centrifuged (3300 x g for 10 min). The organic layer was collected in another glass tube and evaporated in a vacuum centrifuge (Univapo 150H, UniEquip, Martinsried, Germany). The residue was taken up in 250 µL of mobile phase-water (80:20, v/v) and 75 µL n-hexane, and the mixture was transferred in an auto sampler glass vial. After centrifugation (13000 x g for 2 min), the hexane layer was discarded, and 200 µL of the remainder was injected into the HPLC system equipped with a Supelcosil LC-18 column (5 µm, 4.6 mm x 150 mm) for COM1 or Waters XTerra RP 8 column (5 µm, 3.9 x 150 mm) for COM2 and COM3. The column temperature was 40°C, and the absorbance was detected at a wavelength of 312 nm (COM1), 441 nm (COM2), or 261 nm (COM3). The mobile phase (isocratic gradient) consisted of ammonium acetate 10 mM-acetonitrile (45:55, COM1; 50:50, COM2) or ammonium acetate 10 mM-triethylamine 0.1% in acetonitrile (58:42, COM3) and was pumped at a rate of 1.0 mL/min. The peak corresponding to the unchanged compound was collected in a polyethylene vial by a fraction collector (Pharmacia LKB SuperFrac) and analyzed for radioactivity. Concentrations of the test compound in samples were calculated from the ratio of the amount of radioactivity in the eluted fraction and the area of the absorbance of the non-radiolabeled test compound used as internal standard.

3.7 Data analysis

Total radioactivity concentrations, expressed as ng-eq/mL or ng-eq/g, were estimated by dividing the radioactivity concentration in samples by the specific radioactivity of administered test compound using Microsoft Excel. Concentrations of parent drug were determined by the principle of reverse isotope dilution using following equation in Microsoft Excel

ID

AD

IS

AS

A

A

A

A=

where AAS is the amount of analyte in the sample (unknown, to be determined), AIS is the amount of internal standard added to the sample, AAD is the amount of analyte detected, and AID is the amount of internal standard detected. AAD was calculated using R/(SRxS) where R is the amount of radioactivity determined in the peak fraction, SR is the specific radioactivity, and S is the slope. The amount of internal standard detected was calculated as AID=Area/RF-AAD where RF is the response factor (Area/ng). The level of quantification (LOQ) was set to 75 dpm. LOQs of

23

radioactivity and test compound in blood, plasma, urine, and tissues were calculated by dividing 75 dpm by the specific radioactivity of the administered test compound and by the sample amount. P values were calculated with a two-sample t-test in Microsoft Excel assuming unequal variances. The level of significance was set at P 0.90. Volume of distribution (V0) was calculated by dividing the dose by the concentration at time zero (C0). C0 was obtained by extrapolation to zero time of the concentration-time plot in semilogarithmic scale.

24

4 Results and discussions

4.1 Hemolytic activity of excipients

In vitro results

CEL/EtOH 65:35, EtOH, HP-β-CyD, PEG 200, and Solutol did not induce hemolysis in dog and human blood at 0.5% and a contact time of 4 h (data not shown). In contrast, TPGS at 0.5% incubated with blood of various species caused hemolysis in a time-dependent manner (Figure 4-1). Erythrocytes from rat and human were more sensitive than those of mouse and dog, indicated by cell lysis at shorter contact times. Reducing the TPGS concentration from 0.5% to 0.1% induced no hemolysis in all four species in the investigated time range (data not shown).

010

2030

4050

6070

1 h 2 h 4 h 6 h

Incubation time

Hem

oly

sis

(%

)

Rat

Mouse

Dog

Human

Figure 4-1 Effect of incubation time on the hemolytic activity of TPGS

Induced hemolysis by 0.5% TPGS in blood of various species (n=3, mean ± SD). Hemolysis in rat blood after 6-h incubation was not determined.

Discussion

Except for TPGS, all tested excipients (CEL, EtOH, HP-β-CyD, Solutol, and PEG 200) were non-hemolytic which is consistent with data reported in the literature (134,135,136,137,138) and the fact that they are widely used in commercially available parenteralia (54). TPGS at 0.5% exhibited marked hemolysis after longer contact time (>1 h), whereas TPGS at 0.1% showed no hemolysis under equal incubation conditions. The detected hemolysis might possibly result not mainly from TPGS but from metabolites, namely α-tocopheryl succinate and polyethylene glycols, both being able to destruct erythrocytes (134,139,140). This phenomena could contribute to the extensively delayed onset of hemolysis. For the investigations, TPGS at 0.5% was used in the non-hemolytic time range.

25

4.2 Impact of the hematocrit on blood partition parameters

In vitro results

Whole blood derived from three species was incubated with COM2 (100 ng/mL) at varying hematocrit values. Concentrations of COM2 in blood and plasma were measured at equilibrium, and partition parameters calculated from these data are summarized in Table 4-1. Concentrations in blood, plasma, and blood cells remained unaffected by the hematocrit value (0.40-0.60). The partition parameter BPR was also similar over the investigated hematocrit range, whereas BCPR changed slightly and FP distinctly, both decreasing by increasing the hematocrit from 0.40 to 0.60.

Table 4-1 Effect of hematocrit on the in vitro blood distribution of COM2

Blood cell concentrations and partition parameters (FP, BPR, and BCPR) derived from [3H]COM2

concentrations measured in blood and plasma using same blood pools at different hematocrit values (n=3, mean ± SD).

Species Hematocrit Concentration (ng/mL) FP BPR BCPR

Blood Plasma Blood cells (%) Mouse 0.40 50 ± 1 1.21 ± 0.03 1.52 ± 0.07 0.45 46 ± 2 1.20 ± 0.06 1.44 ± 0.13 0.50 101 ± 3 87 ± 4 116 ± 8 43 ± 0 1.16 ± 0.01 1.32 ± 0.01 0.55 40 ± 1 1.13 ± 0.04 1.23 ± 0.06 0.60 36 ± 1 1.12 ± 0.03 1.19 ± 0.05 Dog 0.40 48 ± 2 1.26 ± 0.06 1.66 ± 0.14 0.45 44 ± 1 1.25 ± 0.04 1.57 ± 0.09 0.50 109 ± 4 90 ± 2 131 ± 15 41 ± 1 1.25 ± 0.03 1.51 ± 0.05 0.55 38 ± 1 1.19 ± 0.03 1.35 ± 0.06 0.60 35 ± 1 1.13 ± 0.02 1.22 ± 0.03 Human 0.40 70 ± 3 0.86 ± 0.03 0.66 ± 0.08 0.45 65 ± 2 0.84 ± 0.03 0.65 ± 0.07 0.50 101 ± 5 125 ± 10 78 ± 6 63 ± 2 0.80 ± 0.03 0.60 ± 0.06 0.55 55 ± 2 0.82 ± 0.03 0.68 ± 0.06 0.60 55 ± 1 0.73 ± 0.02 0.55 ± 0.03

Discussion

The in vitro method for investigating distribution of drugs in blood commonly uses whole blood freshly prepared and pooled. Drug concentrations in blood and plasma are determined. Based on these data, further partition parameters, including CBC, FP, BPR, and BCPR, can be estimated, but they are partially dependent on the hematocrit. Therefore, it is important to know how the hematocrit affects these parameters, thereby providing useful information for comparing results. With this in mind, present experiments were performed over the entire physiological hematocrit range in blood pools of three different species (mouse, dog, and human). COM2 was used as test compound due to sufficient availability.

The rank order of hematocrit influences was FP > BCPR > BPR > CB ≈ CP ≈ CBC with most pronounced changes for FP and none for CB/CP/CBC. Parameters calculated from concentrations measured in samples decreased constantly with increasing the hematocrit (0.40-0.60), which was most distinct for FP. But within a hematocrit variation of 0.05 none of the parameters was dependent on the

26

hematocrit. Consequently, blood partition data obtained from in vitro experiments with similar hematocrits are consistent and can be compared together. For data comparison across studies, hematocrit adjusting to values of previous studies is suggested taking into consideration a difference of ≤0.05 between the lowest and highest value.

4.3 Major binding proteins of model compounds

In vitro results

Figure 4-2 illustrates the qualitative binding of model compounds to isolated proteins compared to the total fraction bound in plasma. The following ranking was obtained with regard to decreasing order of protein binding:

COM1: albumin > α1-acid glycoprotein > γ-globulins ≈ lipoproteins; COM2: albumin > lipoproteins > γ-globulins >> α1-acid glycoprotein; COM3: α1-acid glycoprotein > albumin > γ-globulins >> lipoproteins; COM4: albumin > α1-acid glycoprotein >> γ-globulins ≈ lipoproteins; COM5: albumin ≈ α1-acid glycoprotein >> γ-globulins ≈ lipoproteins.

0

10

20

30

40

50

60

70

80

90

100

COM1 COM2 COM3 COM4 COM5

Bo

un

d f

racti

on

(%

)

Plasma Albumin AGP γ-globulins HDL LDL VLDL

Figure 4-2 Qualitative differences in protein binding patterns of model compounds in vitro

Total protein-bound fraction of compounds in human plasma compared to the qualitative extent of compound binding to various isolated human proteins (albumin, AGP, γ-globulins, and lipoproteins such as HDL, LDL, and VLDL). Each bar represents mean ± SD (n=3).

Discussion

In vitro experiments showed the binding of model compounds with different degrees to the three major drug-binding proteins in plasma (albumin, α1-acid glycoprotein, lipoproteins). A high binding to albumin (A) and α1-acid glycoprotein (AGP) was found for COM1 and COM4 (A>AGP), COM5 (A≈AGP), and COM3 (A

27

4.4 The impact of Vitamin E TPGS on COM1 in rat

In vitro results

The equilibrium of COM1 between plasma and blood cells was reached within few minutes (

28

Table 4-3 Comparative plasma kinetics of COM1 with and without TPGS

Plasma concentrations and pharmacokinetic parameters of [3H]COM1 administered intravenously in formulations based on glucose 5% (control) or TPGS 20% to rats (LOQ=2 pg/mL, n=3, mean ± SD). Data are normalized to a dose of 1 µg/kg. AUC(u)0.08-0.5h relates to area under unbound drug plasma concentration-time curve.

Time Control TPGS

(h) COM1 in plasma (pg/mL) Percentage of control value

0.08 184 ± 77 110 ± 6 60 0.25 59 ± 13 39 ± 6 66 0.5 32 ± 11 20 ± 6 62 C0 (pg/mL) 215 132 AUC0.08-0.5h (pg·mL

-1·h) 32 20

AUC(u)0.08-0.5h (pg·mL-1

·h) 0.67 0.76 t1/2 (h) 0.17 0.17 funchanged (%) 18.7 5.5 fu (%) 2.1 ± 0.1 3.8 ± 0.3

Parent drug Metabolites

10

100

1000

0 0.25 0.5

Time (h)

CO

M1

(p

g/m

L)

100

1000

10000

0 0.25 0.5

Time (h)

Rad

ioacti

vit

y (

pg

-eq

/mL

)

Figure 4-3 Influence of TPGS on the systemic exposure of COM1 and metabolites

Plasma concentration-time profiles of parent drug and metabolite-related radioactivity of [3H]COM1 after intravenous administration to rats. COM1 was injected in glucose 5% (closed symbols, black line) or TPGS 20% (open symbols, dashed line). Data shown are normalized to a dose of 1 µg/kg, and symbols represent single values and lines mean values (n=3). Values of metabolite-related radioactivity were obtained by subtracting concentrations of parent drug from concentrations of total radioactivity.

Discussion

Drug-excipient interaction studies showed no direct correlation between the in vitro results and the in vivo situation in rats. Upon intravenous administration of COM1 in TPGS, slightly lower plasma concentrations and binding to plasma proteins were observed in animals as compared to those received COM1 in a TPGS-free solution. In contrast to these in vivo findings, COM1 displayed in vitro no alterations in the presence of TPGS at 0.5%, whereas a higher TPGS concentration (5%) led to

29

enhanced distribution into plasma and a higher fraction bound in plasma. Furthermore, very rapid metabolism and elimination of COM1 under in vivo conditions contributed to a pharmacokinetic profile inappropriate for studying drug-excipient interactions.

4.5 The impact of Vitamin E TPGS on COM2 in mouse

In vitro results

To assess whether the ultracentrifugation time could be shortened for minimizing the degradation of COM2 in plasma (147), blank plasma samples were centrifuged, and time aliquots were analyzed for total protein concentrations in plasma and plasma water. After 4-h centrifugation, protein levels in the plasma water section were below 0.05 ng/mL corresponding to ~0.1% of total plasma proteins adequate to assure a sufficient separation of plasma water and proteins. Therefore, samples were collected after 4-h centrifugation for analysis.

COM2 (0.01-100 ng/mL) distributed almost equally between whole blood and plasma (FP ~45%) and was very highly bound to plasma proteins (>98%, mainly to albumin and lipoproteins) (Figure 4-2). The plasma fraction (63%) was enhanced for COM2 in TPGS at the beginning (

30

(a)

0.0

0.5

1.0

1.5

0.08 h 1 h

BP

R(b)

0.0

0.5

1.0

1.5

2.0

0.08 h 1 h

BC

PR

(c)

0

20

40

60

80

100

0.08 h 1 h

ρ

Figure 4-4 TPGS-mediated alteration of COM2 distribution in blood

Blood-plasma (a), blood cell-plasma (b), and blood cell-unbound in plasma (c) concentration ratios of [3H]COM2 at 0.1 ng/mL in the presence (white bars) and absence (black bars) of TGPS (0.5%) after 0.08-h and 1-h incubation in mouse blood (pH 7.3, H 0.45, n≥3, mean ± SD).

In vivo results

In a previous study (153), the disposition kinetics of COM2 was evaluated in mice after iv administration in a solution containing HP-β-CyD 10% (5 mL/kg). To enable a comparison of former data with those in the current investigation for internal purposes only, COM2 was injected at a volume of 5 mL/kg dissolved in plasma or TPGS 10%. The blood concentration of TPGS from this injection preparation was estimated as ~0.7% assuming a blood volume of 72 mL/kg. Thus, the amount of TPGS in blood was higher in vivo compared to the amount used in vitro (0.5%).

The administration of COM2 as TPGS-containing solution at the same dosage caused approximately 2-fold higher plasma concentrations as compared to COM2 formulated in plasma (control) (Table 4-5, Figure 4-5). These findings are in line with data obtained in blood distribution studies in vitro, where partitioning into blood cells was reduced in the presence of TPGS, resulting in a higher concentration in plasma at the same total blood concentration. Furthermore, in vivo a higher plasma exposure to metabolites and a slower elimination of metabolites from the systemic circulation were found for COM2 in TPGS, indicated by 4-fold increased AUC and t1/2 of metabolite-related radioactivity in plasma (Table 4-6). Determination of unbound COM2 in plasma could not be performed on samples from mice due to concentrations below LOQ (3 pg/mL).

No differences in tissue concentrations were observed between both groups, although COM2 in TPGS resulted in a decrease of V0 (Table 4-5) and KP (Table 4-7), both suggesting altered tissue distribution and being in line with higher drug accumulation in the circulation. Because the free drug fraction in plasma generally reflects more accurately distribution processes due to the ability of unbound drugs to pass through membranes and then reach the target organ, the free drug fraction determined in vitro was considered. Calculated tissue-unbound in plasma concentration ratios, KP(u), were reduced in the TPGS group only within the first minutes after drug administration (Table 4-7).

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Table 4-5 Comparative plasma kinetics of COM2 with and without TPGS

Plasma concentrations (a) and pharmacokinetic parameters (b) of [3H]COM2 after iv dosing at 400 ng/kg in formulations based on blank plasma (control) or TPGS to mice (LOQ=3 pg/mL, n=3, mean ± SD). *,** significantly different from the control at P

32

Table 4-6 Comparative plasma kinetics of metabolites of COM2 with and without TPGS

Plasma concentrations and pharmacokinetic parameters of metabolite-related radioactivity after iv administration of [3H]COM2 (400 ng/kg) in blank plasma (control) or TPGS 10% to mice (n=3, mean ± SD). Values of metabolite-related radioactivity were obtained by subtracting concentrations of parent drug from concentrations of total radioactivity. ** significantly different from the control at P

33

Discussion

Results signified the ability of the excipient TPGS to modify the blood distribution of COM2 in mouse under in vitro and in vivo conditions in a similar manner. Plasma concentrations of COM2 and metabolites were significantly increased, and the free fraction of drug in plasma (in vitro) decreased. Concentrations in tissues were independent of the formulation, whereas distribution ratios of drug in tissue to drug unbound in plasma were lower within the first minutes after dosing COM2 in TPGS. Overall, the altered pharmacokinetic profile of COM2 in plasma suggests drug inclusion in excipient-micelles and/or promoted protein binding by excipient in plasma.

The altered disposition of COM2 and metabolites in plasma is likely caused by the ability of TPGS to form micelles (141). Drug trapping by micelles in blood can be responsible for increased total plasma concentrations and decreased unbound fraction in plasma, thereby influencing drug accumulation in plasma and blood cells (94). Changes in the free fraction could also be caused by altered protein binding. There are different suggested mechanisms by which formulation vehicles can influence the free fraction of compounds, such as vehicle-compound interactions (association and/or micellar encapsulation) and vehicle-protein interactions. The interaction either promotes or blocks the binding of the compound in plasma. Most likely, different interacting processes contribute to the effective free fraction (125).

The alteration in tissue distribution at the beginning may be induced by changes of the free drug fraction in the presence of TPGS and exists only for few minutes probably due to the excipient degradation in blood. The phenomenon found after some minutes post-dose is reported in the literature for Paclitaxel formulated in Cremophor EL (94). The main characteristics are disproportionally increased plasma concentrations accompanied by unchanged tissue levels and tissue distribution processes.

4.6 The impact of hydroxypropyl-β-cyclodextrin on COM3 in rat

In vitro results

COM3 was predominantly located in the cellular fraction (80%) and was moderately bound to plasma proteins with high binding to α1-glycoprotein and albumin in a concentration-independent manner (Table 4-8, Figure 4-2). The drug partitioning into blood cells and the fraction bound to proteins were markedly reduced in incubations containing HP-β-CyD, consequently lowering both the blood-plasma and blood cell-unbound in plasma concentration ratios (Table 4-8, Figure 4-6). Whereas HP-β-CyD decreased the protein binding suggesting more COM3 available for uptake into cells, higher plasma levels associated with reduced concentrations in cells were observed for COM3 in HP-β-CyD. In conclusion, HP-β-CyD was selected for the in vivo study because of the most pronounced drug-excipient interactions detected in vitro.

34

(a)

0

1

2

3

None HP-β-CyD

BP

R(b)

0

1

2

3

4

5

None HP-β-CyD

BC

PR

(c)

0

10

20

30

40

None HP-β-CyD

ρ

Figure 4-6 HP-β-CyD-mediated alteration of COM3 distribution in blood

Blood-plasma (a), blood cell-plasma (b), and blood cell-unbound in plasma (c) concentration ratios of [3H]COM3 at 5 (white bars) and 500 ng/mL (black bars) in the presence and absence of HP-β-CyD (0.5%) after incubation in rat blood (pH 7.6, H 0.44, n≥3, mean ± SD).

Table 4-8 Effect of excipients on blood distribution and protein binding of COM3 in vitro

Partition parameters of [3H]COM3 obtained at equilibrium after incubation with and without excipients (0.5%) in rat blood (pH 7.6, H 0.44, n≥3, mean ± SD).

Excipient COM3 FP BPR BCPR fu ρ

(ng/mL) (%) (%) None 5 20.9 ± 1.5 2.6 ± 0.1 4.6 ± 0.2 12.1 ± 0.9 39.9 ± 3.9 500 19.5 ± 0.3 2.9 ± 0.0 5.3 ± 0.1 12.2 ± 0.2 43.1 ± 0.8

5 19.4 ± 1.4 2.9 ± 0.2 5.6 ± 0.2 nd nd CEL/EtOH, 65:35 (v/v) 500 17.5 ± 0.3 3.2 ± 0.1 6.0 ± 0.1 nd nd HP-β-CyD 5 38.5 ± 2.2 1.4 ± 0.1 2.1 ± 0.2 40.1 ± 1.8 5.1 ± 0.5 500 35.3 ± 0.4 1.6 ± 0.0 2.3 ± 0.0 39.6 ± 0.7 5.9 ± 0.1 Solutol 5 17.4 ± 1.4 3.2 ± 0.3 6.1 ± 0.4 nd nd 500 16.1 ± 0.3 3.5 ± 0.1 6.7 ± 0.1 nd nd PEG 200 5 19.7 ± 2.0 2.9 ± 0.3 5.1 ± 0.6 nd nd 500 18.6 ± 0.5 3.0 ± 0.1 5.6 ± 0.2 nd nd

5 22.7 ± 1.8 2.5 ± 0.2 4.4 ± 0.4 12.6 ± 0.1 34.6 ± 3.6 EtOH/PEG200/Glu, 5:5:90 (v/v/v) 500 20.0 ± 0.4 2.8 ± 0.1 5.1 ± 0.1 11.8 ± 0.2 43.2 ± 1.0 TPGS 5 24.9 ± 1.8 2.3 ± 0.2 4.0 ± 0.2 nd nd 500 23.7 ± 0.3 2.4 ± 0.0 4.1 ± 0.1 nd nd

In vivo results

Since COM3 dissolved in EtOH/PEG200/Glu (5:5:90, v/v/v) showed similar distribution kinetics in vitro as compared to glucose 5% without additives (Table 4-8), the control formulation consisted of EtOH/PEG200/Glu. This assured a sufficient solubility of COM3.

Dosing COM3 in a HP-β-CyD-containing formulation resulted in decreased protein binding and blood cell partitioning as compared to the control group (Ta